Molecular and clinical characterization of a founder mutation causing G6PC3 deficiency

G6PC3 deficiency is a monogenic immunometabolic disorder that causes syndromic congenital neutropenia. Patients display heterogeneous extra-hematological manifestations, contributing to delayed diagnosis. Here, we investigated the origin and functional consequence of the G6PC3 c.210delC variant found in patients of Mexican origin. Based on the shared haplotypes amongst carriers of the c.210delC mutation, we estimated that this variant originated from a founder effect in a common ancestor. Furthermore, by ancestry analysis, we concluded that it originated in the indigenous Mexican population. At the protein level, we showed that this frameshift mutation leads to an aberrant protein expression in overexpression and patient-derived cells. G6PC3 pathology is driven by the intracellular accumulation of the metabolite 1,5-anhydroglucitol-6-phosphate (1,5-AG6P) that inhibits glycolysis. We characterized how the variant c.210delC impacts glycolysis by performing extracellular flux assays on patient-derived cells. When treated with 1,5-anhydroglucitol (1,5-AG), the precursor to 1,5-AG6P, patient-derived cells exhibited markedly reduced engagement of glycolysis. Finally, we compared the clinical presentation of patients with the mutation c.210delC and all other G6PC3 deficient patients reported in the literature to date, and we found that c.210delC carriers display all prominent clinical features observed in prior G6PC3 deficient patients. In conclusion, G6PC3 c.210delC is a loss-of-function mutation that arose from a founder effect in the indigenous Mexican population. These findings may facilitate the diagnosis of additional patients in this geographical area. Moreover, the in vitro 1,5-AG-dependent functional assay used in our study could be employed to assess the pathogenicity of additional G6PC3 variants.


INTRODUCTION
Human glucose-6-phosphatase catalytic subunit 3 (G6PC3) de ciency is an autosomal recessive disorder rst identi ed by Boztug et al. in 2009 as a cause of syndromic severe congenital neutropenia (SCN) 1 .In addition to accelerated apoptosis, neutrophils from patients show impaired chemotaxis and bactericidal activity 2,3 .More than 110 patients have been described in the literature, and the clinical spectrum of G6PC3 de ciency continues to expand.Aside from neutropenia, the most frequently observed features include cardiac defects, urogenital malformations, prominent super cial veins, and endocrine abnormalities [4][5][6] .However, around 20% of reported cases of G6PC3 de ciency manifest as a non-syndromic form of SCN 4,7,8 .Moreover, instances of cyclic neutropenia have been observed sporadically in patients 9,10 .The phenotypic heterogeneity, coupled with the rarity of this disorder, contributes to delays in diagnosis and treatment.
In practice, it is often necessary to rule out several potential diagnoses before suspecting a genetic defect in patients with G6PC3 de ciency, and the process of identifying disease-causing variants through genetic testing afterward is intricate 11 .As most publicly available genetic databases were built using samples from individuals of European descent, alleles speci c to other populations may not be well represented in these datasets 12,13 .Thus, variants observed in patients from underrepresented ancestries are more prone to being classi ed as variants of uncertain signi cance (VUS), adding further complexity in achieving a prompt diagnosis 14 .Furthermore, the absence of an effective functional test to measure the deleteriousness of novel G6PC3 variants leads to additional challenges in establishing disease causality.Here, we describe and functionally characterize a founder mutation in G6PC3 observed in patients from Mexico and of indigenous American ancestry.

Study approval
This study was conducted in accordance with the Helsinki Declaration, with written informed consent obtained from the patients and their families.Approval for this study was obtained from the Vanderbilt University Medical Center Institutional Review Board (IRB), Nashville, USA.

Estimating G6PC3 allele age
Blood-derived DNA collected from 10 carriers of the G6PC3 c.210delC allele (two trios and two motherchild pairs) were sequenced using Illumina short-read sequencing.The affected chromosome (chr17) was sequenced at an average read depth of 10.92X across all ten individuals.Raw reads were aligned to human reference genome build GRCh38/hg38 using Burrows-Wheeler Aligner 15 (0.7.17).PCR duplicates were subsequently identi ed in the aligned reads, and base quality scores were recalibrated following the GATK Best Practices Work ow 16 .Pre-called GVCFs from these six individuals underwent subsequent joint variant calling using the HaplotypeCaller 17 function from GATK (4.2.0.0).Finally, variant quality scores were recalibrated for both SNPs and indels.
The jointly called VCF was pruned using VCFtools 18 (0.1.15)and VcfFilter from the BIOPET suite 19 (0.2).After removing variants with any missingness across the six individuals, those with a minor allele count of less than 3, a quality score of less than 30, or a minimum read depth of less than 20 were also removed.
Using this pruned variant set, the affected chromosome (chr17) was phased using SHAPEIT4 20 (4.2.0), with 3,202 1000 Genomes samples sequenced at 30X 21 serving as the reference panel.The phased VCF was converted to hap/sample format using bcftools 22 (1.12).Using R (4.0.3), the hap le was reformatted as a 13-column text le containing the chr21 coordinate and phased variant calls for each of the 12 phased chromosomes (2 per individual) at that site.Phased variant calls could then be manually spot-checked for concordance with the aligned reads using the IGV genome browser 23 (2.9.4).As genetic data from the fathers were unavailable in two of the four pedigrees, these carrier haplotypes were inferred from the mother and proband haplotypes using a custom Python script.The lengths in centimorgans (cM) of the chr17 chromosomal arms upstream and downstream of the mutation site were calculated, and these respective lengths were used as inputs for the Mutation_age_estimation.R script developed by Gandolfo et al. 24 .A con dence coe cient of 0.95 without chance sharing correction was used, assuming a correlated genealogy.

Ancestry inference using PCA
To determine the genetic ancestries of the G6PC3 c.210delC carriers, we combined the variant calls from 10 samples with those from 2,343 reference samples with African, American, and European ancestries from the harmonized HGDP + 1KGP dataset 25 .Variant ltering was performed according to Hardy-Weinberg equilibrium (P < 1×10 − 6 ) and missingness using PLINK v.1.9 26.Autosomal variants with a minor allele frequency > 1% were retained and pruned for linkage disequilibrium (r 2 = 0.2).PCA was conducted using 157,601 variants with smartpca implemented in EIGENSOFT version 8.0.0 27 .Eigenvectors were calculated using 2,343 reference samples, and the 10 G6PC3 c.210delC carriers were projected onto these eigenvectors.A second PCA was performed using 8,032 variants located on chr17.
After phasing, RFMix (v2.03-r0) 30 was used to estimate local ancestry across chr17 for the ten carriers of the G6PC3 c.210delC allele.

Establishment of Epstein-Barr Virus (EBV)-Immortalized B Cell Lines
For generation of EBV-B cell lines derived from patients and healthy control individuals, puri ed B cells were immortalized with EBV as previously reported 31 .
Plasmid cloning and site-directed mutagenesis RNA was extracted from healthy control EBV-B cells using the RNeasy Plus Mini Kit (Qiagen, Venlo, The Netherlands).RNA was reverse transcribed using the Verso cDNA Synthesis Kit (ThermoFisher Scienti c, Waltham, MA).Primer sequences used to generate the full-length cDNA of G6PC3 with a His-tag right next to the start or stop codon of cDNA that encodes the G6PC3 protein were as follows: N-ter His-tag Forward: 5'-ATG CAT CAC CAT CAC CAT CAC ATG GAG TCC ACG CTG G -3'; N-ter His-tag Reverse: 5'-TCA GGA AGA GTG GAT GGG − 3'; C-ter His-tag Forward: 5'-ATG GAG TCC ACG CTG G -3'; C-ter His-tag Reverse: 5'-TCA GTG ATG GTG ATG GTG ATG GGA AGA GTG GAT GGG C -3'.The products were TAcloned into a pcDNA3.1 plasmid vector using the pcDNA 3.1/V5-His TOPO TA Expression Kit (ThermoFisher Scienti c) according to the manufacturer's protocol.Plasmid DNAs were puri ed from bacterial clones with a Miniprep kit (ThermoFisher Scienti c).Then, constructs carrying the 210delC mutant allele were generated by site-directed mutagenesis with primers: Forward: 5'-CTC AAC CTC ATT TCA AGT GGT T -3'; Reverse: 5'-AAC CAC TTG AAA TGA GGT TGA G -3'.In brief, after the PCR reaction using PfuUltra II Fusion High-delity DNA Polymerase (Agilent, Santa Clara, CA), 1uL DpnI (NEB, Ipswich, MA) was added followed with incubation at 37C for 3 hours.Mutagenesis was validated by Sanger sequencing.

HEK293T cell transfection
In 6-well plates, HEK293T cells were transfected in 2 mL media with 2.5ug of pcDNA3.1 empty vector, N or C-terminal His-tagged WT G6PC3, or N-or C-terminal His-tagged 210delC mutant constructs using Lipofectamine 3000 Transfection Reagent (ThermoFisher Scienti c) according to the manufacturer's protocol for 48 hours.

G6PC3 mRNA detection by RT-qPCR
Total RNA was extracted from transfected HEK293T cells and EBV-B cells using the RNeasy Kit (Qiagen, Venlo, The Netherlands).The RT-qPCR was performed using the Luna Universal One-Step RT-qPCR Kit (NEB) on the CFX96 RT-qPCR detection system (Bio-Rad, Hercules, CA).The following primers were used to amplify the cDNA of G6PC3: Forward: 5'-TCA AGT GGT TTC TTT TTG GAG -' 3; Reverse: 5'-ATC ATG CAG TGT CCA GAA G -' 3. The G6PC3 mRNA expression level in each sample was normalized to the expression level of the GUS gene transcript.

The membrane protein fraction of EBV-B cells was obtained with the Mem-PER Plus Membrane Protein
Extraction Kit (ThermoFisher Scienti c).The membrane protein extracts were mixed with loading buffer, incubated at 37°C for 30 minutes, and subjected to electrophoresis in a precast 4%-20% gradient gel (Bio-Rad).

Extracellular ux assay
EBV-B cells were pretreated with 2mM of 2-DG (Cayman Chemical, Ann Arbor, MI) or 1,5-AG (Cayman Chemical) for ve days prior to the glycolysis stress assay.Cells were counted and resuspended in Agilent Seahorse XF RPMI 1640 medium supplemented with 2mM glutamine, with 2mM of 2-DG or 1,5-AG added to corresponding conditioned cells.150,000 live cells/well were plated.ECAR was measured on a Seahorse XFe 96 bioanalyzer using the glycolysis stress test with sequential injections of 100mM glucose, 15µM oligomycin, and 500mM 2-DG.Glycolysis rate was quanti ed as the change in ECAR before and after addition of glucose.Glycolytic capacity was calculated as the difference between maximal ECAR reached following the oligomycin injection and the non-glycolytic acidi cation prior to addition of glucose.

RESULTS
The G6PC3 c.210delC variant found in Mexico originated from a founder effect Among all the mutations causing G6PC3 de ciency, a few are only found in speci c ethnic groups, implying founder effects 6 .For instance, the c.210delC variant has been reported in 13 G6PC3 de cient patients, who are either homozygous or compound heterozygous for this variant 4,[32][33][34] .Among them, 12 patients are of Mexican descent, while another patient is included from the North American Severe Chronic Neutropenia International Registry 32 .These observations led us to interrogate whether a founder effect causes the recurrence of this mutation or if it is a mutational hotspot.To study this, we recruited four patients from central Mexico who are homozygous for this variant and were born to unrelated, nonconsanguineous parents (Fig. 1A-B).We collected genomic DNA from these four patients plus six of their heterozygous healthy parents.We performed whole-genome sequencing (WGS) followed by haplotype analysis on the region of chromosome 17 surrounding G6PC3.Since samples were unavailable from 2 of the patients' fathers, these two carrier haplotypes were inferred computationally from the mother and patient haplotypes.Our analysis revealed a shared haplotype segment surrounding the mutation site among all carriers (Fig. 1C).The length of this shared haplotype suggests that the mutation originated 26 generations ago (95% con dence interval, 2.3-50.5) in a common ancestor.This analysis indicates that the G6PC3 c.210delC single-nucleotide deletion is a founder mutation in Mexico.

The G6PC3 c.210delC mutation is of Indigenous American origin
Based on the assumption that each generation interval is between 20 and 25 years, the estimated age of the G6PC3 c.210delC variant is 520-650 years.This allele age raised the question of whether the shared haplotype originated from native American ancestry or was introduced to Mexico by Europeans.To investigate this, we conducted principal component analysis (PCA) with WGS data of 10 carriers, using reference populations from the combined 1000 Genomes Project (1KGP) and Human Genome Diversity Project (HGDP) dataset.Ancestry inference analysis on the whole genome and chromosome 17 showed that the carriers of this mutation cluster closely with the indigenous American populations from HGDP (Fig. 2A-B).Moreover, we used 2,343 deeply sequenced reference samples of individuals of European, African, and Ad Mixed American Ancestry from the 1KGP + HGDP dataset to perform local ancestry estimation across chr17.Our analysis indicates that the chromosomal region containing the G6PC3 c.210delC mutation (chr17: 44,071,175) is of American origin (Fig. 2C).Overall, our analysis shows that the mutation c.210delC originated in the indigenous Mexican population.The G6PC3 c.210delC variant results in reduced G6PC3 mRNA and complete loss of protein expression G6PC3 encodes a protein with nine transmembrane domains that localizes to the endoplasmic reticulum 35,36 .The c.210delC variant observed in our patients is a single-nucleotide deletion in exon 1 of G6PC3 (Fig. 3A).It is predicted to cause a shift in the reading frame after amino acid 70 (in the second transmembrane domain of the protein), thus introducing a premature stop codon 46 amino acids after the mutation site (p.F71Sfs*46) (Fig. 3B).To examine the impact of this variant at mRNA and protein levels, we rst transfected human embryonic kidney 293T (HEK293T) cells with plasmids containing Cor N-terminally Histidine-tagged versions of wildtype (WT) and p.F71Sfs*46 G6PC3.RT-qPCR results suggest that the mutation induces a moderate reduction in G6PC3 mRNA expression (Fig. 3C).Western blotting of whole cell lysate with an anti-His-tag antibody yielded a 10kDa band in the cells transfected with N-terminal tagged p.F71Sfs*46 G6PC3, aligning with the anticipated molecular weight of a truncated mutant protein (Fig. 3D).This predicted mutant protein would not retain enzymatic activity as it lacks the active site of G6PC3, which is composed of amino acids R79, H114, and H167 36 (Fig. 3B).By analyzing the C-terminal His-tagged mutant G6PC3, we showed that there is no translation re-initiation after the premature termination codon (Fig. 3D).To gain additional insights in a more physiologically relevant context, we utilized Epstein-Barr Virus immortalized B (EBV-B) cells derived from two patients and healthy controls.We observed markedly reduced levels of G6PC3 mRNA, potentially due to nonsense-mediated mRNA decay (Fig. 3E).Furthermore, using membrane protein fractions extracted from EBV-B cells for western blotting and using a polyclonal antibody with epitope spanning the Nterminal region of human G6PC3, we showed that neither the WT nor the mutant size protein was expressed in cells from the patients (Fig. 3F).Altogether, our data demonstrate that the G6PC3 c.210delC (p.F71Sfs*46) variant disrupts mRNA and protein levels, causing a complete loss of expression.
Cells from patients with the G6PC3 c.210delC variant show abolished G6PC3 enzymatic function G6PC3 is a metabolite-repair enzyme involved in the hydrolysis of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), which is the phosphorylated form of a food-derived polyol named 1,5-anhydroglucitol (1,5-AG) 37 .When a functional G6PC3 is absent, 1,5-AG6P can accumulate to a concentration that inhibits hexokinase activity 37 .Accumulation of 1,5-AG6P thereby impairs glycolysis since hexokinase mediates the rate-limiting rst step of the glycolytic pathway 38 .This mechanism leads to neutropenia since neutrophils rely heavily on glycolysis to ful ll their energetic needs 39 .To directly assess the metabolic consequence of the absence of G6PC3 caused by the c.210delC variant, we used patient-derived EBV-B cells to determine if they have defective hexokinase activity in mediating glycolysis.EBV-B cells express all four isoforms of hexokinase (I, II, III, and ADP-glucokinase) identi ed in mammalian cells, thus permitting us to test the functional impact of the G6PC3 de ciency (Fig. S1).EBV-B cells from patients and healthy controls were treated with either 1,5-AG or glycolytic inhibitor 2-Deoxy-D-Glucose (2-DG) for ve days.Then, we quanti ed the glycolytic activities of these cells through measurements of extracellular acidi cation rate (ECAR).Patient EBV-B cells exhibited similar levels of glycolytic activity to cells derived from healthy controls, whether untreated or treated with 2-DG.However, patient cells showed signi cantly impaired glycolysis rate and glycolytic capacity following the 1,5-AG treatment, while healthy control cells remained unaffected (Fig. 4A-C).These results illustrate the metabolic disturbance in cells from patients with the G6PC3 c.210delC variant due to the absence of G6PC3, establishing ECAR measurements in EBV-B cells as a means to examine the functional consequences of G6PC3 mutations.
Patients with the G6PC3 c.210delC variant show a clinical pro le similar to other G6PC3-de cient patients As all patients with the G6PC3 c.210delC variant are from the same geographical area, we evaluated whether they exhibit any characteristic in their clinical presentation that may differentiate them from the rest of the reported G6PC3 de cient patients denoting some environmental aspects of the disease.To this end, we collected the clinical information from all published cases of G6PC3 de ciency to compare the frequency of appearance of nine prominent clinical features between patients with and without the G6PC3 c.210delC mutation (n = 14, some published, some unpublished) (Fig. 5).None of these patients showed isolated neutropenia but present with features of syndromic severe congenital neutropenia including extra-hematological abnormalities.Except for hepatosplenomegaly, all other features of G6PC3 de ciency have been observed in these patients.We also noticed that patients with the c.210delC variant display a higher occurrence of thrombocytopenia, endocrine abnormalities, and hearing loss.Although this may imply a speci c characteristic of this group of patients, it might also re ect some variability in standard clinical testing.Overall, our analysis shows that patients who are carriers of the G6PC3 c.210delC mutation display all main clinical characteristics described in G6PC3 de ciency, indicating that the mutation may be the main driver of their disease.

DISCUSSION
G6PC3 de ciency is a rare genetic disorder with a broad phenotypic spectrum, posing di culties for timely diagnosis.The differential diagnosis process can be particularly complicated for patients with non-syndromic neutropenia or less frequently observed clinical features [40][41][42] .Mutations observed in G6PC3 de cient patients spread across all six exons of the gene 6 .Interestingly, the prevalence of several G6PC3 alleles varies signi cantly amongst different ethnic groups.These include the p.Phe44Ser mutation of Pakistani origin and the p.W73X mutation in patients from the Dominican Republic 43,44 .
Here, through haplotype analysis and ancestry inference, we demonstrated that the G6PC3 c.210delC variant was recurrently observed in Mexico due to a founder effect, and it is of native American origin.
We also reviewed the signs and symptoms in patients with this variant, concluding that they closely resemble those observed in other reported patients.These ndings may facilitate targeted testing of patients from this region with unexplained congenital neutropenia.
The mechanisms responsible for the phenotypic variability in G6CP3 de ciency remain elusive thus far 6,8, 45 .It has been hypothesized that these variations might be correlated with the residual G6PC3 enzymatic activity resulting from some mutations 46 .To assess the functional impact of mutations, previous studies measured the capacity of mutant G6PC3 in mediating hydrolysis of glucose-6phosphate (G6P) using microsomes isolated from transfected yeast or COS-1 cells 1,46 .However, it has been recently established that the primary physiological role of G6PC3 is not to dephosphorylate G6P into glucose and phosphate.Instead, the molecular mechanism underlying neutrophil dysfunction observed in G6PC3 de cient patients is associated with the accumulation of 1,5-AG6P 37,47 .This highlights the need for a 1,5-AG-dependent functional test to assess the pathogenicity of G6PC3 variants.Using Seahorse extracellular ux assays, we have shown that EBV-B cells from our patients fail to eliminate 1,5-AG6P, leading to defective glycolytic activity, indicating that the G6PC3 c.210delC variant disrupts the metabolite repair activity of G6PC3.One limitation is that we utilize immortalized cells rather than primary cells from patients to illustrate this effect.Nevertheless, this in vitro assay could still be employed in future studies to determine the impact of G6PC3 mutations in additional patients, which would aid in establishing potential genotype-phenotype correlations for this disease, as well as predicting the pathogenicity of VUS.
In conclusion, this study identi ed the G6PC3 c.210delC allele as a founder mutation that abolishes protein expression and function.These ndings may help expedite the diagnosis of G6PC3 de ciency, especially in the Mexican population.As previous reports suggest that G6PC3 de ciency can lead to death from severe infections when neutropenia is left untreated, prompt diagnosis and provision of treatments are critical 48,49 .Importantly, empagli ozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor frequently used to treat type 2 diabetes, has successfully resolved neutrophil defects in patients with G6PC3 de ciency by lowering the 1,5-AG blood concentrations 47,50,51 .Along with the use of this highly effective, safe, and easy-to-take oral alternative to granulocyte-colony-stimulating factor (G-CSF) injections, early disease diagnosis may improve outcomes of G6PC3 de cient patients

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DeclarationsAUTHORSHIP CONTRIBUTIONSXZ and RMB wrote the manuscript.ERG, JCR, YI, JM, SOLR, and RMB conceptualized the study.XZ, MJB, MEK, and ARP conducted experiments, data collection, and analysis.EAMT, SCSM, DAHS, GLH, YS, OMM, POR, and SOLR collected patient samples and characterized patient clinical information.All authors reviewed and approved the nal manuscript.